| Objective: To investigate the mechanisms resulting aberrance of regulatory cells subtypes in patients with Kawasaki Disease.Methods: Forty-eight children with KD and twenty-four age-matched health controls were consented to participate in this study. 1. Flow cytometry was performed to evaluate the proportions of IL-35+Breg, Treg and expression levels of programmed death ligand 1(PD-L1), CD169, programmed death 1(PD-1), CD43, IL-12p35, IL-27EBI3, IL-12Rβ2, IL-27Rα, phosphated signal transducer and activator oftranscription 1(p STAT1), p STAT3, IL-10, transforming growth factor bata(TGF-β), cytotoxic T lymphocyte-associated antigen 4(CTLA4), STIP1 homology and U-Box containing protein 1(STUB1), heat shouck protein 70(HSP70), and ubiquitin-specific-processing protease 7(USP7). 2. Transcription levels of SHP-2, phosphatase and tensin homolog(PTEN), Vav, IL-1R1, IL-1RAP, IL-6Rα, glycoprotein 130(gp130), TNFR1, My D88 and receptor interacting serine/threonine protein kinase 1(RIP1) were detected by quantitative real-time PCR. 3. Chrosomal-Immunoprecipitation and Western-Blot was used to determine the poly-ubiquitination status of Foxp3 protein. 4. Plasma concentrations of IL-1β, IL-6, IL-12, IL-35 and TNF-α were measured by enzyme-linked immunosorbent assay.Results: 1. The proportions of IL-35+Breg and its expressions of IL-12p35, IL-27EBI3 and IL-10 inpatients during acute KD were found to be lower than those of healthy controls(P<0.05), and restored to some extent after treatment(P<0.05). 2. The proportion of Treg and levels of IL-12p35, IL-27EBI3, IL-10, TGF-beta, CTLA4 and Foxp3 were down-regulated during acute KD(P<0.05),as polyubiquitination of Foxp3 protein was significantly enchanced. All the items mentioned before increased significantly after treatment. A positive correlation was found between the proportion of Treg and IL-35+ Breg in patients with acute KD(r=0.69,P<0.05). Meanwhile, the proportion of Treg and expression levels of IL-10, TGF-beta, CTLA4 and Foxp3 in KD patients with coronary artery lesions(KD-CAL+) were lower than those without coronary artery lesions(KD-CAL-)while poly-ubiquitination level of Foxp3 protein in KD-CAL+ group was much higher than that in KD-CAL- group(P<0.05), and restored to certain extent.upon treatment(P<0.05). 3. Compared with healthy controls, plasma concentrations of IL-35 and expression levels of IL-12Rβ2, IL-27Rα, p STAT1 and p STAT3 in CD19+ B cells were significantly down-regulated during acute KD, as plasma concentrations of IL-12,TNF-α and expression levels of PD-L1 and CD169 in CD14+ B cells were elevated remarkably(P<0.05).Hower, all the items mentioned before restored after treatment(P<0.05). 4. Expression levels of USP7 in CD4+T cells were decrease significantly during acute KD while the levels of STUB1 and HSP70 and the ratio of STUB1/USP7 were elevated. In addition, there is a negative correlation between STUB1/USP7 ratio and protein level of Foxp3 found in patients with acute KD(r=-0.56, P<0.05).Compared with KD-CAL-group, expressions of STUB1 and HSP70 and the ratio of STUB1/USP7 increased a lower expression of USP7 was found in KD-CAL+group(P<0.05). 5. Plasma concentrations of inflammatory cytokines(IL-1β, IL-6, and TNF-α) were up-regulated during acute KD while expression levels of surface receptors(IL-1R1/IL-1RAP/TLR4, IL-6Rα/gp130 and TNFR1) and its downstream molecules(My D88, p STAT3 and RIP1) in CD4+T cells were up-regulated(P<0.05), and restored remarkably after therapy(P<0.05). Simultaneously, all the items mentioned before in KD-CAL+ group were found to be higher than those in KD-CAL- group(P<0.05). No differences about other TLRs were found among the groups(P>0.05).Conclusion: Insufficiency and derangement of IL-35+ Breg may be one of the important factors contributing to immunological dysfunction in Kawasaki disease, and hyper-ubiquitination of Foxp3 protein maybe involved in the mechanism resulting aberrant Treg in KD. |
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